Cytochrome oxidase (COX), the terminal complex of the respiratory chain, consists of 11 different subunit polypeptides. The mitochondrially encoded Cox1p, Cox2p and Cox3p subunits constitute the catalytic core while eight other structural subunits are products of nuclear genes. Because of its genetic and compositional complexity, biogenesis of COX is a highly integrated process that is assisted by numerous accessory proteins and regulatory factors for coordinating a balanced output of compartmentally separated genes. Pulse-chase studies with isolated mitochondria have shown that prior to their assimilation into COX, Cox1p and Cox3p, each, transitions through a series of intermediates differentiated by their compositions of accessory factors and structural subunits. This finding has led us to propose a biogenesis model involving three separate pathways with modular products that ultimately combine to form the holoenzyme. This proposal has four objectives, each related to an important facet of COX biogenesis. 1) We wish to consolidate the modular assembly model with direct experimental evidence that Cox2p also preassembles as an independent module. This will be addressed by identifying and characterizing Cox2p intermediates in pulse-chase labeled mitochondria. 2) We have indirect evidence that mitochondrial gene products of COX and the bc1 complex, both of which exist in a supercomplex, are inserted in the same or neighboring subcompartments of the membrane during their biogenesis. This suggests that assembly of the COX/bc1 supercomplex may be coordinated both physically and temporally. We will test this hypothesis by genetic and biochemical means. 3) Some recent results suggest that the proper stoichiometry of COX and ATP synthase may be achieved via a complex of the rotor subunit Atp9p and the COX peripheral subunit Cox6p. We will further test this interesting possibility by studying assembly of the ATP synthase in cox6 null mutants and conversely of COX assembly in atp9 mutants. 4) Much of our current knowledge of COX biogenesis has come from functional studies of accessory factors that intervene at different stages of the Cox1p-Cox3p assembly pathways. Many but not all of these factors were identified through studies of yeast mutants displaying a specific deficiency in COX. The fourth and last objective of this proposal is to continue mining a collection of nuclear respiratory defective yeast pet mutants for still undiscovered genes essential for COX biogenesis. These studies will enlarge our understanding of the mechanisms and the factors that govern assembly of an important mitochondrial respiratory complex derived from genetic information residing in two spatially distinct compartments of the cell.